ASTM D4012-15

Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water

SIGNIFICANCE AND USE
5.1 A rapid and routine procedure for determining biomass of the living microorganisms in cultures, waters, wastewaters, and in plankton and periphyton samples taken from surface waters is frequently of vital importance. However, classical techniques such as direct microscope counts, turbidity, organic chemical analyses, cell tagging, and plate counts are expensive, time-consuming, or tend to underestimate total numbers. In addition, some of these methods do not distinguish between living and nonliving cells.  
5.2 This test method measures the concentration of cellular-ATP present in the sample. ATP is a constituent of all living cells, including bacteria, algae, protozoa, and fungi. Consequently, the presence of cellular-ATP is an indicator of total metabolically active microbial contamination in water. ATP is not associated with matter of non-biological origin.  
5.3 The ATP (luciferin-luciferase) method is a rapid, sensitive determination of viable microbial biomass. ATP is the primary energy donor for life processes, does not exist in association with nonliving detrital material, and the amount of ATP per unit of biomass (expressed in weight) is relatively constant. (ATP per cell varies with species and physiological state of the organism.)  
5.4 This test method can be used to:  
5.4.1 Estimate viable microbial biomass in cultures, waters, and wastewaters.  
5.4.2 Estimate the amount of total viable biomass in plankton and periphyton samples.  
5.4.3 Estimate the number of viable cells in a unispecies culture if the cATP content (or if the average amount of cATP) per cell is known.  
5.4.4 Estimate and differentiate between zooplanktonic, phytoplanktonic, bacterial, and fungal cATP through size fractionation of water, and wastewater samples.  
5.4.5 Measure the mortality rate of microorganisms in toxicity tests in entrainment studies, and in other situations where populations or assemblages of microorganisms are placed under stress.  
5.5 This test m...
SCOPE
1.1 This test method covers a protocol for capturing, extracting and quantifying the cellular adenosine triphosphate (cATP) content associated with microorganisms normally found in laboratory cultures, waters, wastewaters, and in plankton and periphyton samples from waters.  
1.2 The ATP is measured using a bioluminescence enzyme assay, whereby light is generated in amounts proportional to the concentration of ATP in the samples. The light is produced and measured quantitatively as relative light units (RLU) which are converted by comparison with an ATP standard and computation to pg ATP/mL.  
1.3 This method does not remove all known chemical interferences, known to either luminesce in the 530 ± 20 nm range, or to quench light emitted in that range. It should not be used to determine ATP concentrations in samples with dissolved organic compounds, heavy metals or >10 000 ppm total dissolved solids. Alternative methods have been developed for determining ATP concentrations in fluids samples likely to contain such interferences (Test Methods D7687 and E2694).  
1.4 Knowledge of the concentration of ATP can be related to viable biomass or metabolic activity of microorganisms (Appendix X1).  
1.5 This test method offers a high degree of sensitivity, rapidity, accuracy, and reproducibility.  
1.6 The analyst should be aware that the precision statement pertains only to determinations in reagent water and not necessarily in the matrix being tested.  
1.7 This test method is equally suitable for use in the laboratory or field.  
1.8 The method normally detects cATP concentrations in the range of 0.1 pg cATP/mL (–1.0Log10 [pg cATP/mL]) to 4 000 000 pg cATP/mL (6.6 Log10 [pg cATP/mL]) in 50 mL water samples.  
1.9 Providing interferences can be overcome, bioluminescence is a reliable and proven method for qualifying and quantifying ATP, although the method does not differentiate between ATP from different sources, for exa...

General Information

• Status: Historical
• Publication Date: 30-Jun-2015
• ICS: 13.060.50 - Examination of water for chemical substances
• Technical Committee: D19 - Water
• Drafting Committee: D19.24 - Water Microbiology

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D4012 − 15
Standard Test Method for
Adenosine Triphosphate (ATP) Content of Microorganisms
1
in Water
This standard is issued under the fixed designation D4012; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 1.9 Providing interferences can be overcome, biolumines-
cence is a reliable and proven method for qualifying and
1.1 This test method covers a protocol for capturing, ex-
quantifying ATP, although the method does not differentiate
tracting and quantifying the cellular adenosine triphosphate
between ATP from different sources, for example, from differ-
(cATP) content associated with microorganisms normally
ent types of microorganisms, such as bacteria, fungi, algae and
found in laboratory cultures, waters, wastewaters, and in
protozoa.
plankton and periphyton samples from waters.
1.10 The values stated in SI units are to be regarded as
1.2 The ATP is measured using a bioluminescence enzyme
standard. No other units of measurement are included in this
assay, whereby light is generated in amounts proportional to
standard.
the concentration of ATP in the samples. The light is produced
and measured quantitatively as relative light units (RLU) 1.11 This standard does not purport to address all of the
which are converted by comparison with an ATP standard and
safety concerns, if any, associated with its use. It is the
computation to pg ATP/mL. responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
1.3 This method does not remove all known chemical
bility of regulatory limitations prior to use.
interferences, known to either luminesce in the 530 6 20 nm
range, or to quench light emitted in that range. It should not be
2. Referenced Documents
used to determine ATP concentrations in samples with dis-
2
solved organic compounds, heavy metals or >10 000 ppm total
2.1 ASTM Standards:
dissolved solids. Alternative methods have been developed for
D1129 Terminology Relating to Water
determining ATP concentrations in fluids samples likely to
D1193 Specification for Reagent Water
contain such interferences (Test Methods D7687 and E2694).
D1601 Test Method for Dilute Solution Viscosity of Ethyl-
ene Polymers
1.4 Knowledge of the concentration of ATP can be related to
D4175 Terminology Relating to Petroleum Products, Liquid
viable biomass or metabolic activity of microorganisms (Ap-
Fuels, and Lubricants
pendix X1).
D5847 Practice for Writing Quality Control Specifications
1.5 This test method offers a high degree of sensitivity,
for Standard Test Methods for Water Analysis
rapidity, accuracy, and reproducibility.
D6161 Terminology Used for Microfiltration, Ultrafiltration,
1.6 The analyst should be aware that the precision statement
Nanofiltration and Reverse Osmosis Membrane Processes
pertains only to determinations in reagent water and not
D6300 Practice for Determination of Precision and Bias
necessarily in the matrix being tested.
Data for Use in Test Methods for Petroleum Products and
Lubricants
1.7 This test method is equally suitable for use in the
D7687 Test Method for Measurement of Cellular Adenosine
laboratory or field.
Triphosphate in Fuel, Fuel/Water Mixtures, and Fuel-
1.8 The method normally detects cATP concentrations in the
Associated Water with Sample Concentration by Filtration
range of 0.1 pg cATP/mL (–1.0Log [pg cATP/mL]) to 4 000
10
E2694 Test Method for Measurement of Adenosine Triphos-
000 pg cATP/mL (6.6 Log [pg cATP/mL]) in 50 mL water
10
phate in Water-Miscible Metalworking Fluids
samples.
F1671 Test Method for Resistance of Materials Used in
1
This test method is under the jurisdiction of ASTM Committee D19 on Water
2
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved July 1, 2015. Published September 2015. Originally contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
approved in 1981. Last previous edition approved in 2009 as D4012 – 81 (2009). Standards volume information, refer to the standard’s Document Summary page on
DOI: 10.1520/D4012-15. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

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D4012 − 15
Protective Clothing to Penetration by Blood-Borne Patho- 3.1.14 lysis, n—disintegration or destruction of whole bac-
gens Using Phi-X174 Bacteriophage Penetration as a Test terial cells. F1671
System
3.1
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D4012 − 81 (Reapproved 2009) D4012 − 15
Standard Test Method for
Adenosine Triphosphate (ATP) Content of Microorganisms
1
in Water
This standard is issued under the fixed designation D4012; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This test method covers the measurement of adenosine triphosphate (ATP) in microorganisms in concentrationsa protocol
for capturing, extracting and quantifying the cellular adenosine triphosphate (cATP) content associated with microorganisms
normally found in laboratory cultures, waters, wastewaters, and in plankton and periphyton samples from waters.
1.2 The ATP is measured using a bioluminescence enzyme assay, whereby light is generated in amounts proportional to the
concentration of ATP in the samples. The light is produced and measured quantitatively as relative light units (RLU) which are
converted by comparison with an ATP standard and computation to pg ATP/mL.
1.3 This method does not remove all known chemical interferences, known to either luminesce in the 530 6 20 nm range, or
to quench light emitted in that range. It should not be used to determine ATP concentrations in samples with dissolved organic
compounds, heavy metals or >10 000 ppm total dissolved solids. Alternative methods have been developed for determining ATP
concentrations in fluids samples likely to contain such interferences (Test Methods D7687 and E2694).
1.4 Knowledge of the concentration of ATP can be related to viable biomass or metabolic activity, or by utilizing an average
concentration (or amount) of ATP per cell, an estimated count of microorganisms can activity of microorganisms (Appendix X1be
obtained in the case of unispecies cultures.).
1.5 This test method offers a high degree of sensitivity, rapidity, accuracy, and reproducibility. However, extreme care must be
taken at each step in the analysis to ensure meaningful and reliable results.
1.6 The analyst should be aware that the precision statement pertains only to determinations in reagent water and not necessarily
in the matrix being tested.
1.7 This test method is equally suitable for use in the laboratory or field.
1.8 The method normally detects cATP concentrations in the range of 0.1 pg cATP/mL (–1.0Log [pg cATP/mL]) to 4 000 000
10
pg cATP/mL (6.6 Log [pg cATP/mL]) in 50 mL water samples.
10
1.9 Providing interferences can be overcome, bioluminescence is a reliable and proven method for qualifying and quantifying
ATP, although the method does not differentiate between ATP from different sources, for example, from different types of
microorganisms, such as bacteria, fungi, algae and protozoa.
1.10 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.11 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory
limitations prior to use.
2. Referenced Documents
2
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D1601 Test Method for Dilute Solution Viscosity of Ethylene Polymers
1
This test method is under the jurisdiction of ASTM Committee D19 on Water and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved May 1, 2009July 1, 2015. Published June 2009September 2015. Originally approved in 1981. Last previous edition approved in 20022009 as
D4012 – 81 (2002).(2009). DOI: 10.1520/D4012-81R09.10.1520/D4012-15.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
D4012 − 15
D4175 Terminology Relating to Petroleum, Petroleum Products, and Lubricants
D5847 Practice for Writing Quality Control Specifications for Standard Test Methods for Water Analysis
D6161 Terminology Used for Microfiltration, Ultrafiltration, Nanofilt
...